The Genome Responses And MTF-1 Regulation Mechanism To Heavy Metal Stress In Oyster

In recent years, the sea area heavy metal pollution is becoming serious. Oyster living in intertidal area could highly accumulate heavy metal without being poisoning. It was widely used for heavy metal pollution detection. For heavy metal accumulation research in oyster, previous studies mainly foucused on their physiological mechanisms, while there are few studies focused on the molecular mechanisms. In our research, for one hand, we used the genome comparision and transcriptome data analysis to screen oyster heavy metal responsive genes and explained oyster expanded defensin genes and pathways as the main reason for their heavy metal accumulation. For the other hand, we have conducted the transcriptional factor functional research using cloning, protein prokaryotic expression, RNA interference and electrophoretic mobility shift assay(EMSA) analysis. We explained the MTF-1 regulation mechanisms in oysters. Also we studied the functions of MTF-1 regulated genes for oyster heavy metal defense. The following were the main conclusions:1. Transcriptome analysis under heavy metal stress(1) The physiology experiment indicated that oyster accumulated Cd and Pb, and not for Cu, Zn and Hg in our treated conditions. Analysis the responsive genes numbers under different heavy metal stress, we found that there are more downregulated genes in RNA-seq database. Heavy metal tend to inhibite the genes expressions. MMC cluster analysis of the whole gene set revealed the responsive mechanism of oyster under heavy metal stress. Many heavy metal responsive genes have paralog differentiation and these paralog displayed expression differentiations which may help oyster adapt to diversity stress environment.(2) The principle component analysis of oyster gene sets revealed these genes displayed tissues and times expression differentiation. The gills mainly played important roles after 12 h treatment while the digestvie gland mainly function after 9 d treatment.(3) The transcriptome analysis of Zn, Cd and Zn+Cd transcriptome data revealed the oysterresponsive mechanims under these stress. Also, we found that both of Zn and Cd challenge decreased the Zn or Cd accumulation in oysters.2. Analysis heavy metal responsive gene families and metabolism pathwaysFirst, we conducted gene family analysis. We analyzed the oyster expanded gene family CYP450 and found that the clan 2 tended to expanded and displayed tandem duplication. Metallothionein have two subfamilies in oyster. There are clustered MRE motifs in MTI promoter regions, which indicated that MTF-1 may have more powerful regulated abilities for it. The three MTI genes have the similar expression patterns. Their affinities for heavy metal ions were: Cd>Hg>Zn>Pb>Cu。Second, we conducted metabolism pathways analysis. After heavy metal treated by 12 h, the glutathione and phytochelatins synthesis related genes were up-regulated which indicated that they played important roles for oyster heavy metal first defense line. Also CYP450 metabolism pathway and ABC transporter pathway were enriched under heavy metal stress, indicating their important roles for heavy metal defense. Third, according to genome comparison and RNA-seq analysis, we identified the heavy metal responsive genes, including oxidative responsive, heavy metal conjugative, antioxidant, transporters and chelation. These genes displayed expanded tendency compared with other species. Also, oyster expanded KOs co-expressed in five metabolism aspects, which also played important roles in oyster heavy metal defense. The KEGG maps analysis in different species also revealed that these pathways expanded in oysters. The oyster expanded genes families and metabolism pathways revealed oyster heavy metal adaptations.3. The transcriptional mechanism of MTF-1 geneWe cloned the oyster heavy metal transcriptional factor MTF-1 and conducted the functional experiments. The results indicated that MTF-1 mainly expressed in oyster nucleus under normal condition. The EMSA experiment revealed that MTF-1 expressed protein could bind to the MRE probes and regulated the other genes. We conducted RNAi to found the oyster heavy metal responsive genes regulated by MTF-1. The MTF-1 expression was inhibited more than seventy five percents. Its down-regulated genes Zn T1, MTI and MTIV expressions were also down-regulatedafter MTF-1 expression was inhibited. We cloned MTI and MTIV genes upstream 3K sequences, and the result indicated that the MRE clustered in MTI upstream while there was only one MRE element in MTIV gene. These clustered MRE may help the MTF-1 had stronger regulatory capacity for MTI gene. These may help to explain that the MTI expressed highly than MTIV under heavy metal stress.4. The heavy metal Zn accumulation mechanismIn order to explain the Zn accumulation mechanism, we analyzed the oyster zinc responsive genes. The results indicated that MTI genes increased 36 and 1.9 folds higher than the control, while the Zn T1 genes increased 11.9 and 52 folds higher than the control. This indicated that Zn T1 gene played more important roles for oyster zinc transport after 12 h and 9 d treatment. The MTI gene may play more important role for oyster oxidative responsive after 12 h treatment.